The long-term goal of this proposal is to develop a method for heritable, cell-specific knock down of gene expression in the nematode C. elegans. The ability to knock down gene expression in individual cell types in this organism will allow a more detailed understanding of protein function and will permit the dissection of cellular interactions such as those present in neural circuits. Our strategy has several advantages over current knock down methods and it usurps the nonsense-mediated decay (NMD) machinery that is present in all eukaryotic cells designed to degrade mRNA transcripts containing premature termination codons. In our strategy we express the gene of interest at endogenous levels with a degradation tag - a 3'-untranslated region (3'-UTR) that targets the mRNA transcript for degradation in wild-type cells but not in cells in which the NMD machinery has been compromised by mutation. Using cell-specific promoters we then convert NMD- deficient cells into NMD-competent cells to cause cell-specific degradation of mRNA transcripts containing the degradation tag. To provide proof that our method will work, we provide preliminary results in which we have fused a heterologous NMD degradation tag onto a reporter gene and show NMD-dependent knock down of reporter mRNA expression. We also show that we can knock down the function of an endogenous C. elegans gene using this heterologous degradation tag to cause behavioral defects similar to those observed in null mutants. In this proposal we plan to replace the heterologous degradation tag used in pilot studies with gene- specific degradation tags that will preserve the gene's 3'-UTR necessary for normal expression and stability. Thus in this proposal we plan to: 1) Develop and test gene-specific degradation tags for knock down and use them to knock down expression of endogenous genes in specific cell types while preserving wild-type expression in all other cells; 2) Quantitate transgene expression and knock down efficiency; and 3) compare the efficiency of our knock down method with the other heritable method (hairpin RNA, hpRNA) using unc-4 (a gene largely refractory to knock down by all other methods) as our target gene. Unlike other methods of gene knock down, our strategy does not rely on RNA interference and thus there is absolutely no spreading of the knock down effects to other cells. Our strategy can knock down the expression of any gene in any cell type and thus will likely be adopted by most C. elegans researchers. Because many of the proteins expressed in C. elegans are homologous to proteins expressed in humans (~40% of all proteins) we expect that the analysis of protein function in C. elegans using our cell-specific knock down strategy will shed new light on the physiological function of homologous proteins in humans. PUBLIC HEALTH RELEVANCE: C. elegans has proven to be a valuable genetic model organism for study of important biological processes including neurogenesis and degeneration, developmental programming and cell death. Many of the molecular mechanisms identified in the worm that control and/or mediate these processes are conserved in humans and thus studies in C. elegans have contributed to our understanding of many human pathological conditions. The ability to stably and heritably knock down gene function in individual cells in C. elegans will allw unprecedented insight into the cell-specific function of proteins. The knock down approach described in this proposal can be used to study the function of any protein in any cell type to understand any biological process including those that directly benefit human health such as neurotransmission, muscle function, and cell division.